It is often necessary, for health and safety reasons, to detect small amounts of substances in biological and environmental samples. The detection of such substances would ideally be done with good sensitivity and specificity, easily, and at low cost. Such detection is often difficult, however, due to a variety of reasons, including the complexity of the samples that are being tested.
Chloramphenicol (CAP) is an antibiotic used for the treatment of bacterial infections, and it is often administered to animals for disease prevention (M. E. Falagas, A. P. Grammatikos, A. Michalopoulos, Potential of old-generation antibiotics to address current need for new antibiotics, Expert Review of Anti-Infective Therapy, 6 (2008) 593-600; J. A. Turton, C. M. Andrews, A. C. Havard, T. C. Williams, Studies on the haemotoxicity of chloramphenicol succinate in the Dunkin, Hartley guinea pig, International Journal of Experimental Pathology, 83 (2002) 225-238). CAP is a protein synthesis inhibitor that acts primarily by binding reversibly to the 505 ribosomal subunit and also can inhibit mitochondrial protein synthesis in mammalian cells. Therefore, despite its role in disease prevention and nonproliferation of bacterial growth, CAP is also associated with potentially serious toxic effects in humans, including bone marrow depression.
Moreover, antibiotic residues in animal milk may cause allergic reactions or lead to antimicrobial resistance. The use of CAP in food animals is, therefore, illegal in most countries, including the USA, Canada, China, and members of the European Union. However, the illicit use of CAP in cows to control mastitis and other animal diseases continues because of its low cost and easy availability (G. Y. Liu, C. Y. Chai, Towards the development of a sensitive electrochemical sensor for the determination of chloramphenicol residues in milk, Analytical Methods, 7 (2015) 1572-1577).
Analysis of ultra-trace level of contaminants, including CAP, in complex biological sample matrices is a daunting challenge due to the presence of numerous potential interferents in these samples.
Solid-phase extraction (SPE) is considered the gold standard among conventional sample preparation techniques, routinely used for the pre-concentration and clean-up of the target analyte(s) from complex sample matrices for subsequent analysis (B. Buszewski, M. Szultka, Past, Present, and Future of Solid Phase Extraction: A Review, Critical Reviews in Analytical Chemistry, 42 (2012) 198-213). However, conventional silica-based sorbents (e.g., C8, C18, etc.) do not offer adequate selectivity and specificity because the target analytes are predominantly retained on these sorbents by non-specific hydrophobic interactions, leading to simultaneous co-extraction of numerous endogenous interfering substances from the sample, thereby complicating the subsequent chromatographic analysis.
Molecularly imprinted polymers (MIPs) are synthetic polymeric materials that possess specific cavities complimentary to the shape, size, and functional groups of a template molecule used in the imprinting process (Techniques and Instrumentation in Analytical Chemistry, in: S. Börje (Ed.) Techniques and Instrumentation in Analytical Chemistry, Elsevier 2001, pp. ii; P. Manesiotis, L. Fitzhenry, G. Theodoridis, P. Jandera, 4.20—Applications of SPE-MIP in the Field of Food Analysis, in: J. Pawliszyn (Ed.) Comprehensive Sampling and Sample Preparation, Academic Press, Oxford, 2012, pp. 457-471; L. Chen, S. Xu, J. Li, Recent advances in molecular imprinting technology: current status, challenges and highlighted applications, Chemical Society Reviews, 40 (2011) 2922-2942).
Among many different synthesis pathways that can be used to create MIPs, the organic synthesis route appears to be the most popular (W. J. Cheong, S. H. Yang, F. Ali, Molecular imprinted polymers for separation science: A review of reviews, Journal of Separation Science, 36 (2013) 609-628). However, despite the advantages of organically synthesized MIPs, these materials often suffer from significant shortcomings, which include: (a) slow mass transfer kinetics; (b) heterogeneity of the binding sites; (c) low population of high-affinity binding sites; (d) irreversible shrinking and/or swelling when exposed to organic solvents, leading to considerable deformation of the imprinted cavities, and subsequent loss in template recognition capacity; (e) poor extraction performance when the sample matrices are aqueous or biological in nature and the target analytes are polar; (f) lack of ability to imprint thermal- and photo-sensitive template molecules due to the relatively high synthesis reaction temperature; (g) limited template removal option due to low thermal stability of organic polymers; and (h) low imprinting factor (IF) due to relatively high non-specific adsorption.
Some of these shortcomings of organic MIPs have been addressed by sol-gel synthesis approaches. Sol-gel synthesis of MIPs is versatile and possesses advantages including, for example, mild room temperature synthesis conditions, controllable pore size; high surface area, and tunable polarity of the matrix via manipulations in the sol-gel processing conditions such as, for example, the type and concentration of the precursors, catalysts, porogenic agents, and water content. The rigid, highly cross-linked structure of sol-gel MIPs possesses delicately imprinted sites with a high degree of selectivity compared to more flexible organic polymer MIPs.
Despite the potential of sol-gel organic-inorganic hybrid polymers as a host for efficient molecular imprinting, the advantages of these unique material systems have not been fully exploited. This is, in part, due to the lack of thorough understanding of sol-gel chemistry and the involvement of a large numbers of independent variables that eventually determine the ultimate physicochemical characteristics of the sol-gel materials. Irrational optimization of these variables often leads to sol-gel materials with poor accessibility to interaction sites, slow mass transfer rate, ineffective removal of the template, and low adsorption capacity.
Some researchers have proposed surface molecularly imprinted polymers (SMIP) using preformed silica particles as the imprinting host; however, due to the presence of a large number of residual surface silanol groups left on the silica substrate following the molecular imprinting, this approach often leads to high non-specific adsorption and results in low IF (J. Li, M. Yang, D. Huo, C. Hou, X. Li, G. Wang, D. Feng, Molecularly imprinted polymers on the surface of silica microspheres via sol-gel method for the selective extraction of streptomycin in aqueous samples, Journal of Separation Science, 36 (2013) 1142-1148; Y.-M. Yin, Y.-P. Chen, X.-F. Wang, Y. Liu, H.-L. Liu, M.-X. Xie, Dummy molecularly imprinted polymers on silica particles for selective solid-phase extraction of tetrabromobisphenol A from water samples, Journal of Chromatography A, 1220 (2012) 7-13; J.-H. Hu, T. Feng, W.-L. Li, H. Zhai, Y. Liu, L.-Y. Wang, C.-L. Hu, M.-X. Xie, Surface molecularly imprinted polymers with synthetic dummy template for simultaneously selective recognition of nine phthalate esters, Journal of Chromatography A, 1330 (2014) 6-13). As such, surface imprinting is not a viable solution for molecular imprinting if high IF, fast mass transfer kinetic, and high template adsorption capacity are desired.
Milk is a complicated sample matrix that requires multi-step sample preparation procedures. For the isolation of CAP in milk, various techniques have been proposed including liquid-liquid extraction (LLE) (X. Z. Shi, A. B. Wu, S. L. Zheng, R. X. Li, D. B. Zhang, Molecularly imprinted polymer microspheres for solid-phase extraction of chloramphenicol residues in foods, Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 850 (2007) 24-30), solid phase extraction (SPE) (R. S. Nicolich, E. Werneck-Barroso, M. A. S. Marques, Food safety evaluation: Detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry, Analytica Chimica Acta, 565 (2006) 97-102; E. G. Karageorgou, V. F. Samanidou, Development and validation according to European Union Decision 2002/657/EC of an HPLC-DAD method for milk multi-residue analysis of penicillins and amphenicols based on dispersive extraction by QuEChERS in MSPD format, Journal of Separation Science, 34 (2011) 1893-1901; M. Ramos, A. Aranda, M. M. de Pozuelo, T. Reuvers, Chloramphenicol residues in food samples: Their analysis and stability during storage, Journal of Liquid Chromatography & Related Technologies, 26 (2003) 2535-2549), and fabric phase sorptive extraction (FPSE) (V. Samanidou, L. D. Galanopoulos, A. Kabir, K. G. Furton, Fast extraction of amphenicols residues from raw milk using novel fabric phase sorptive extraction followed by high-performance liquid chromatography-diode array detection, Analytica Chimica Acta, 855 (2015) 41-50). Deproteinization of milk is generally used prior to sample enrichment and clean-up by continuous solid phase extraction (D. R. Rezende, N. Fleury Filho, G. L. Rocha, Simultaneous determination of chloramphenicol and florfenicol in liquid milk, milk powder and bovine muscle by LC-MS/MS, Food Additives and Contaminants Part a—Chemistry Analysis Control Exposure & Risk Assessment, 29 (2012) 559-570).
The use of MIPs for the extraction and quantification of CAP in milk-based matrices has been suggested by Mohamed et al. in 2007 (R. Mohamed, J. Richoz-Payot, E. Gremaud, P. Mottier, E. Yilmaz, J. C. Tabet, P. A. Guy, Advantages of molecularly imprinted polymers LC-ESI-MS/MS for the selective extraction and quantification of chloramphenicol in milk-based matrixes. Comparison with a classical sample preparation, Analytical Chemistry, 79 (2007) 9557-9565). An improved method for trace analysis of CAP in honey, urine, milk and plasma using MIPs was proposed by Boyd et al., 2007 (B. Boyd, H. Bjork, J. Billing, O. Shimelis, S. Axelsson, M. Leonora, E. Yilmaz, Development of an improved method for trace analysis of chloramphenicol using molecularly imprinted polymers, Journal of Chromatography A, 1174 (2007) 63-71). The selective determination of CAP at trace levels in milk samples by an electrode modified with molecularly imprinted polymer has also been reported (T. Alizadeh, M. R. Ganjali, M. Zare, P. Norouzi, Selective determination of chloramphenicol at trace level in milk samples by the electrode modified with molecularly imprinted polymer, Food Chemistry, 130 (2012) 1108-1114). CAP was also identified in urine, feed water, milk and honey samples using molecular imprinted polymer clean-up by a commercially available MIPSPE column (Supel MIP) prior to GC/MS analysis after silylation of the antibiotic (M. Rejtharova, L. Rejthar, Determination of chloramphenicol in urine, feed water, milk, and honey samples using MIP clean-up, Journal of Chromatography A, 1216 (2009) 8246-8253). Although Supel MIP columns, presumably synthesized via an organic polymer approach, have demonstrated clear advantages over C18 SPE cartridges in extracting and pre-concentrating CAP from milk and other aqueous samples, due to the simultaneous extraction of non-specific matrix interferents from complex sample matrices, a series of washing steps had to be incorporated into the sample preparation regime followed by vacuum drying of the sorbent and subsequent elution of the analyte with larger volume of eluent.
As such, despite the inherent advantages over C18 SPE sorbent, the CAP imprinted Supel MIP method is labor-intensive, time-consuming and, contradictory to the principle of green analytical chemistry (GAC) (M. Farre, S. Perez, C. Goncalves, M. F. Alpendurada, D. Barcelo, Green analytical chemistry in the determination of organic pollutants in the aquatic environment, Trac-Trends in Analytical Chemistry, 29 (2010) 1347-1362; M. de la Guardia, Green analytical chemistry, Trac-Trends in Analytical Chemistry, 29 (2010) 577-577). Unavoidable application of solvent evaporation followed by sample reconstitution as an integral part of this sample preparation strategy often leads to irreversible analyte loss, poor data quality and low sample throughput.
No sol-gel derived CAP-imprinted sorbent material system has been reported for the separation and detection of this important analyte from milk or other biological samples.